This invention relates to a process for the production of humanized chimera antibody. In contrast to mouse monoclonal antibody, humanized chimera antibody does not cause formation of anti-mouse immunoglobulin antibody in the body of a patient. Thus, side effects are reduced or eliminated and half life in blood increases when the chimera antibody is used. Therapeutic effects which are superior to those obtained in the case of using mouse monoclonal antibody can be obtained in the treatment of human cancers and the like.
It is known that, when mouse antibodies are administered to humans, they are recognized as foreign substances and cause formation of anti-mouse immunoglobulin antibodies in the human body, and the thus formed antibodies react with the administered mouse antibodies. As a result, side effects occur (J. Clin. Oncol., 2, 881 (1984); Blood, 65, 1349-1363 (1985); J. Natl. Cancer Inst., 80, 932 (1988); Proc. Natl. Acad. Sci. U.S.A., 82, 1242 (1985)), the antibodies are cleared away quickly (J. Nucl. Med., 26, 1011 (1985); Blood, 65, 1349-1363 (1985); J. Natl. Cancer Inst., 80, 937 (1988)) and effects of the antibodies are reduced (J. Immunol., 135, 1530 (1985); Cancer Res., 46, 6489 (1986)). When mouse monoclonal antibody is converted into humanized chimera antibody, human anti-mouse immunoglobulin antibody form in minimal amounts if at all, and the half life of the chimera antibody in human blood is six times as long as that of mouse monoclonal antibody (Proc. Natl. Acad. Sci. U.S.A., 86, 4220 (1989)). In addition, it is probable that the Fc region of mouse antibody does not fully activate human complement and human effector cells, in comparison with the Fc region of human antibody. For example, the antitumor activity of mouse monoclonal antibody to ganglioside GD2, which is effected via human effector cells, is improved when the monoclonal antibody is converted into chimera antibody that has the human antibody Fc region (J. Immunol., 144, 1382-1386 (1990)).
Ganglioside is one of the animal cell membrane-constituting glycolipids and is composed of a sugar chain as a hydrophilic side chain, sphingosine as a hydrophobic side chain and fatty acids. It is known that expression of ganglioside varies depending on the type of cells, organs and animal species. In addition, it has been revealed that quantity and quality of the expressed ganglioside change during the canceration process of cells (Cancer Res., 45, 2405 (1985)). For example, it has been reported that gangliosides GD2, GD3, GM2 and the like which hardly exist in normal cells were found in the cells of neuroblastoma, lung small cell carcinoma and melanoma belonging to neuroectodermal-origin tumor which is said to be highly malignant (J. Exp. Med., 155, 1133 (1982); J. Biol. Chem., 257, 12752 (1982); Cancer Res., 47, 225 (1987); ibid., 47, 1098 (1987); ibid., 45, 2642 (1985); Proc. Natl. Acad. Sci. U.S.A., 80, 5392 (1983)).
Ganglioside GD3 has been found most frequently in melanoma cells among the neuroectodermal-origin tumors, and anti-ganglioside GD3 monoclonal antibodies (to be referred to as xe2x80x9canti-GD3 monoclonal antibodyxe2x80x9d hereinafter) belonging to the mouse IgM class and IgG class have been reported (Int. J. Cancer, 29, 269 (1982); J. Biol. Chem., 257, 12752 (1982); Cancer Res., 47, 225 (1987); Acta Neuropathol., 79, 317 (1989); Proc. Natl. Acad. Sci. U.S.A., 77, 6114 (1980); J. Exp. Med., 155, 1133 (1982); Proc. Natl. Acad. Sci. U.S.A., 81, 5767 (1984)).
KM-641 (FERM BP-3116) disclosed in EP-A-0 493 686 is an anti-GD3 monoclonal antibody belonging to the mouse IgG3 class, which reacts not only with ganglioside GD3 but also with ganglioside 3xe2x80x2,8xe2x80x2-LD1 and is possessed of a broad range of antitumor spectrum. In addition, KM-641 has stronger binding activities to antigens than anti-GD3 monoclonal antibody R24 which has been disclosed in J. Exp. Med., 155, 1133 (1982) and it shows strong antitumor activities.
The mouse monoclonal antibody R24 to the ganglioside GD3 was once used for the treatment of melanoma, but the administered mouse monoclonal antibody R24 did not fully exert its effect due to the formation of anti-mouse immunoglobulin antibody in the patient""s body (Eur. J. Cancer Clin. Oncol., 24, suppl 2, s 65 (1988)).
Consequently, the use of chimera antibody for anti-GD3 monoclonal antibody would be advantageous in that anti-mouse immunoglobulin antibody does not form in the body, side effects are reduced or eliminated, its half life in blood is prolonged and its antitumor effector effect increases, and thus therapeutic effects of the chimera antibody which are superior to those of mouse monoclonal antibody can be obtained in the treatment of human cancers and the like.
Several processes for the production of humanized chimera antibodies are known. Humanized chimera antibody, in which constant regions of the heavy chain (to be referred to as xe2x80x9cH chainxe2x80x9d hereinafter) and the light chain (to be referred to as xe2x80x9cL chainxe2x80x9d hereinafter) of mouse monoclonal antibody are converted into human constant regions, is produced in animal cells making use of recombinant DNA techniques. Examples of such processes include a process in which humanized chimera antibody is produced using chromosomal DNA as a gene which encodes mouse H chain variable region (to be referred to as xe2x80x9cVHxe2x80x9d hereinafter) and L chain variable region (to be referred to as xe2x80x9cVLxe2x80x9d hereinafter) (Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, 6851 (1984); Neuberger et al., Nature, 314, 268 (1985); Nishimura et al., Cancer Res., 47, 999 (1987); Dorai et al., J. Immunol., 139, 4232 (1987); Kameyama et al., FEBS letter, 244, 301 (1989)) and another process in which humanized chimera antibody is produced using cDNA (Gillies et al., J. Immunol. Methods, 125, 191 (1989); Liu et al., published International Application in Japan No. 2-501886). Cloning and base sequence determination of hybridoma cell chromosomal DNA which encodes mouse VH and VL require much time and labor in comparison with those of cDNA that encodes mouse VH and VL. Consequently, the process in which cDNA is used for the production of humanized chimera antibody is more desirable than the chromosomal DNA process.
Gillies et al. have succeeded in expressing humanized chimera antibody in animal cells, making use of an expression vector for animal cells having inserted therein a humanized chimera H chain gene obtained by linking mouse VH-encoding cDNA with human CH-encoding chromosomal DNA, and a humanized chimera L chain gene obtained by linking mouse VL-encoding cDNA with human CL-encoding chromosomal DNA (J. Immunol. Methods, 125, 191 (1989)). However, when an attempt was made to prepare chimera antibodies from several types of antibodies, a problem was found that there were certain chimera antibodies whose L chains could not be expressed without converting leader sequences. In addition, humanized chimera antibody can be produced more simply when cDNA which encodes human CH and CL is used instead of the human CH- and CL-encoding chromosomal DNA.
In published International Application in Japan No. 2-501886, Liu et al. discloses a process for the expression of humanized chimera antibody in animal cells, which comprises using an expression vector for animal cells having inserted therein a chimera H chain cDNA obtained by linking mouse VH-encoding cDNA with human CH-encoding cDNA and a chimera L chain cDNA obtained by linking mouse VsL-encoding cDNA with human CL-encoding cDNA. According to this process, however, it is necessary to alter the Ja portion of the VH-encoding cDNA and the JL portion of the VL-encoding cDNA by means of mutation, because the cDNA which encodes mouse VH or VL is linked with the human CH- or CL-encoding cDNA at the J region in the mouse variable region. In addition, with regard to the chimera L chain prepared using mouse Jk5, leucine which is one of the amino acids of the framework 4 is changed to isoleucine when made into humanized chimera antibody. Although amino acid sequence of complementarity-determining region (to be referred to as xe2x80x9cCDRxe2x80x9d hereinafter) is especially important for antigen-antibody binding, the amino acid sequence of the framework is also an important factor. For example, Riechmann et al. have prepared CDR graft antibody by grafting a rat antibody CDR into a human antibody framework and reported that binding activity of the antibody was reduced by the framework conversion and the antibody activity increased when amino acid sequence of the framework was partially changed (Nature, 332, 323 (1988)). Consequently, there is a possibility that the binding activity of humanized chimera antibody is undesirably reduced when the antibody is produced by the mouse Jk5-aided process disclosed by Liu et al.
In view of the above, when any mouse antibody is converted into humanized chimera antibody, it has been desired to simply and easily produce humanized chimera antibody in which amino acids of the mouse antibody variable region remain completely unchanged.
An object of the present invention is to provide a process for the production of humanized chimera antibody by which the chimera antibody is produced easily without changing any of the amino acids of its mouse antibody variable region. Another object of the present invention is to provide a humanized chimera antibody to ganglioside GD3 and a process for the production of such antibody.
The present invention relates to a process for producing humanized chimera antibody which comprises the steps of:
(1) constructing a cassette vector by inserting a cDNA coding for human antibody CH into an expression vector for animal cell use and establishing a cloning site in the upstream region CH of said cassette vector for inserting a cDNA which encodes nonhuman animal VH;
(2) digesting a cDNA coding for nonhuman animal antibody VH with restriction enzymes;
(3) inserting said cDNA coding for nonhuman animal antibody VH into the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5xe2x80x2-end side of said human antibody CH and a base sequence corresponding to the 3xe2x80x2-end side of said nonhuman animal antibody VH and is possessed of restriction enzyme recognition sites on both of its ends, thereby constructing a humanized chimera antibody H chain expression vector in which said cDNA coding for human antibody CH and said cDNA coding for nonhuman animal antibody VH are linked together through said synthetic DNA;
(4) constructing a cassette vector by inserting a cDNA coding for human antibody CL into an expression vector for animal cell use and establishing a cloning site in the upstream region of the CL of said cassette vector for inserting a cDNA which encodes nonhuman animal antibody VL;
(5) digesting a cDNA coding for nonhuman animal antibody VL with restriction enzymes;
(6) inserting said cDNA coding for nonhuman animal antibody VL into the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5xe2x80x2-end side of said human antibody CL and a base sequence corresponding to the 3xe2x80x2-end side of said nonhuman animal antibody VL and is possessed of restriction enzyme recognition sites on both of its ends, thereby constructing a humanized chimera antibody L chain expression vector in watch said cDNA coding for human antibody CL and said cDNA coding for nonhuman animal antibody VL are linked together through said synthetic DNA;
(7) introducing these expression vectors into host cells to obtain a transformant; and
(8) culturing said transformant in an appropriate culture medium, thereby allowing the transformant to produce and accumulate a humanized chimera antibody, and collecting said humanized chimera antibody from the resulting culture broth.
The cassette vector to be used in the present invention is a vector which is obtained by inserting a cDNA that encodes a constant region of human antibody into an expression vector for animal cell use, in which a cloning site is located in the upstream region of the constant region for inserting a cDNA that encodes a variable region of nonhuman animal antibody. An expression vector for humanized chimera antibody can be constructed easily by inserting a variable region of nonhuman animal antibody into the cloning site of the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5xe2x80x2-end side of a constant region of human antibody and a base sequence corresponding to the 3xe2x80x2-end side of a variable region of nonhuman animal antibody and is possessed of restriction enzyme recognition sites on its both ends.
The present invention also relates to a humanized chimera antibody obtainable by the above-described process, a pharmaceutical composition comprising the humanized chimera antibody and a pharmaceutically acceptable carrier, and a method of treating cancer which comprises administering to a patient a pharmaceutically acceptable amount of said humanized chimera antibody.